Nootkatone—a biotechnological challenge

Due to its pleasant grapefruit-like aroma and various further interesting molecular characteristics, (+)-nootkatone represents a highly sought-after specialty chemical. (+)-Nootkatone is accumulated in its producer plants in trace amounts only, and the demand of the food, cosmetics and pharmaceutical industry is currently predominantly met by chemical syntheses. These typically require environmentally critical reagents, catalysts and solvents, and the final product must not be marketed as a “natural flavour” compound. Both the market pull and the technological push have thus inspired biotechnologists to open up more attractive routes towards natural (+)-nootkatone. The multifaceted approaches for the de novo biosynthesis or the biotransformation of the precursor (+)-valencene to (+)-nootkatone are reviewed. Whole-cell systems of bacteria, filamentous fungi and plants, cell extracts or purified enzymes have been employed. A prominent biocatalytic route is the allylic oxidation of (+)-valencene. It allows the production of natural (+)-nootkatone in high yields under mild reaction conditions. The first sequence data of (+)-valencene-converting activities have just become known.

β-Thalassemia mutations in Singapore — a strategy for prenatal diagnosis

The strategy for early prenatal diagnosis of -thalassemia in Singapore by direct detection of the mutant -globin gene requires the spectrum of mutations producing the disorder in this population to be characterized. We analyzed 134 -thalassemia alleles from Singapore by specific oligonucleotide hybridization after DNA amplification, using a nonradioactive enhanced chemiluminescence detection system. The mutations were identified in 90% of the alleles using five oligonucleotide probes for the following mutations: codons 41/42 (deletion -TCTT), IVS II nt 654 (CT), codon 17 (AT), IVS I nt 5 (GC), and -28 TATA box (AG). Together with the strategy of direct sequencing, a total of 97% of the mutations were identified. In the Chinese subpopulation, 97% of the mutations were detected by the oligonucleotide probes. Using just four oligonucleotide probes would identify 96% of the mutations, and 76% of the mutations were accounted for by codon 41/42 (-TCTT) and IVS II nt 654 (CT) mutations. Thus in this subpopulation early prenatal diagnosis would be possible in virtually all the affected families.     

LIMS — beyond CSV: a strategy for LIMS data quality assurance

Computer system validation of a LIMS is often confused with the ongoing task of entering and quality-assuring the material, instrument, specification, method, calculation, and study data required for laboratory sample and results management. A practical approach to LIMS template data quality assurance includes preparation of one or more standard operating procedures for entry and quality assurance of material specifications, test limits, secondary calculation formulae, and other analytical laboratory testing information.     

Membrane-mediated interactions – a physico-chemical basis for protein sorting

Abstract

Sorting of membrane proteins in eukaryotic cells is a complex yet vital task that involves several 10,000 molecular players. Sorting takes place not only along the early secretory pathway, i.e., between the endoplasmic reticulum and the Golgi apparatus, but also between other organelles, including exchange with the cell's plasma membrane. Traditionally, specific binary interactions between proteins have been made responsible for most of the protein sorting. A more active role of lipids, however, became visible in recent years. Not only do lipids in complex membranes show domain formation that may support/suppress sorting events, but also collective, membrane-mediated interactions have emerged as a robust physico-chemical mechanism to drive protein sorting. Here, we will review recent insights into these aspects.     

Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate

Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett–Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on γ-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box–Behnken design. The average γ-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.     

Is there a basis for novel pharmacotherapy of autism?

No medication has yet been shown to consistently alter the symptoms or the course of autism in the majority of patients. The present pharmacotherapy is mainly palliative and sometimes effective in attenuating specific behaviors. The search for better treatment involves examination of the underlying pathophysiology, the genetic or environmental etiology (including possible iatrogenic causes), and assessment of the clinically-generated evidence of efficacy, including serendipitous or unexplained findings. Subtle neuroanatomic and neurochemical changes are being explored and there are anecdotal reports or limited clinical trials that suggest some therapy might be possible. Secretin is a surprising recent addition to the list of candidates. The pharmacologic mechanism by which these agents might provide such effect is not clear, but hypotheses are beginning to emerge. In addition, the prevention of some uncertain number of autism cases is being investigated by examination of certain vaccinations as putative causative or contributory factors. These topics are reviewed in this article, which has the additional purpose of stimulating novel drug discovery efforts for this enigmatic disorder.     

Are white-rot fungi a real biotechnological option for the improvement of environmental health?

The use of white-rot fungi as a biotechnological tool for cleaning the environment of recalcitrant pollutants has been under evaluation for several years. However, it is still not possible to find sufficiently detailed investigations of this subject to conclude that these fungi can decontaminate the environment. In the present review, we have summarized and discussed evidence about the potential of white-rot fungi to degrade such pollutants as polycyclic aromatic hydrocarbons, dyes or antibiotics as an example of the complex structures that these microorganisms can attack. This review also discusses field experiment results and limitations of white-rot fungi trials from contaminated sites. Moreover, the use of catabolic potential of white-rot fungi in biopurification systems (biobeds) is also discussed. The current status and future perspectives of white-rot fungi, as a viable biotechnological alternative for improvement of environmental health are noted.     

Evidence for a genetic basis for the class A Thy-1− defect

When Thy-1^– cell lines derived from different Thy-1⁺ murine thymic lymphomas are analyzed by complementation analysis, most fall into the A complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1⁺ hybrid cell lines between complementary Class A and Class C Thy-1^– mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1⁺ to Thy-1^–. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1^– phenotype was attributable to loss of the gene complementing the Class A Thy-1^– mutation.Other hybrid clones, however, had some thioguanine resistant lines which remained Thy-1⁺. The degree of concordance was a characteristic of the particular hybrid clone examined and subclones which showed complete concordance could be derived from clones showing incomplete concordance. The variability in the degree of concordance between 6-thioguanine resistance and the Thy-1^– phenotype in different hybrid cell lines was also seen among individual hybrid clones isolated from a fusion between a Class A mutant and normal spleen cell blasts.We conclude from these results that the basis of the Class A Thy-1^– phenotype is genetic, but given the variability in the degree of linkage observed, we cannot determine whether the gene determining the Class A mutant phenotype is X-linked in the normal situation.     

In-silico wear prediction for knee replacements—methodology and corroboration

The capability to predict in-vivo wear of knee replacements is a valuable pre-clinical analysis tool for implant designers. Traditionally, time-consuming experimental tests provided the principal means of investigating wear. Today, computational models offer an alternative. However, the validity of these models has not been demonstrated across a range of designs and test conditions, and several different formulas are in contention for estimating wear rates, limiting confidence in the predictive power of these in-silico models.This study collates and retrospectively simulates a wide range of experimental wear tests using fast rigid-body computational models with extant wear prediction algorithms, to assess the performance of current in-silico wear prediction tools.The number of tests corroborated gives a broader, more general assessment of the performance of these wear-prediction tools, and provides better estimates of the wear ‘constants’ used in computational models. High-speed rigid-body modelling allows a range of alternative algorithms to be evaluated. Whilst most cross-shear (CS)-based models perform comparably, the ‘A/A+B’ wear model appears to offer the best predictive power amongst existing wear algorithms. However, the range and variability of experimental data leaves considerable uncertainty in the results. More experimental data with reduced variability and more detailed reporting of studies will be necessary to corroborate these models with greater confidence. With simulation times reduced to only a few minutes, these models are ideally suited to large-volume ‘design of experiment’ or probabilistic studies (which are essential if pre-clinical assessment tools are to begin addressing the degree of variation observed clinically and in explanted components).     

Management strategy evaluation: a powerful tool for conservation?

The poor management of natural resources has led in many cases to the decline and extirpation of populations. Recent advances in fisheries science could revolutionize management of harvested stocks by evaluating management scenarios in a virtual world by including stakeholders and by assessing its robustness to uncertainty. These advances have been synthesized into a framework, management strategy evaluation (MSE), which has hitherto not been used in terrestrial conservation. We review the potential of MSE to transform terrestrial conservation, emphasizing that the behavior of individual harvesters must be included because harvester compliance with management rules has been a major challenge in conservation. Incorporating resource user decision-making required to make MSEs relevant to terrestrial conservation will also advance fisheries science.     

Evaluation of the grazer–prey interaction as a biotechnological strategy to increase toxin production by dinoflagellate cultures in photobioreactors

In this paper, we extend an existing approach to biotechnologically assess grazer–prey interactions between the crustacean Artemia salina (grazer) and the toxic dinoflagellate Protoceratium reticulatum (prey). The applied strategy is presented as a bioprocessing tool for enhancing the production of toxins and bioactive compounds in dinoflagellate cultures. Interactions were based on direct and indirect contact between the grazer and the prey, as well as on the use of different extracts from A. salina cysts and supernatants from cultures in which A. salina had been grown. Several treatments were found to stimulate the growth and yessotoxin production of P. reticulatum mainly due to the action of dissolved excreted substances and/or metabolites released and/or extracted from A. salina. One of the best results was obtained with a culture medium formulation containing 10 % (v/v) supernatant from a culture of A. salina nauplii. This treatment was scaled up to a 15-L photobioreactor. The average maximum specific growth rate (μ _max) of P. reticulatum in this photobioreactor, operated in batch mode, increased by 27 %, whereas the maximum cell concentration (C _max) decreased by 20 % relative to the corresponding control culture. An average increase in yessotoxin production of 50 % with respect to the control culture was observed.     

Exploring a coarse-grained distributive strategy for finite-difference Poisson–Boltzmann calculations

We have implemented and evaluated a coarse-grained distributive method for finite-difference Poisson–Boltzmann (FDPB) calculations of large biomolecular systems. This method is based on the electrostatic focusing principle of decomposing a large fine-grid FDPB calculation into multiple independent FDPB calculations, each of which focuses on only a small and a specific portion (block) of the large fine grid. We first analyzed the impact of the focusing approximation upon the accuracy of the numerical reaction field energies and found that a reasonable relative accuracy of 10⁻³ can be achieved when the buffering space is set to be 16 grid points and the block dimension is set to be at least (1/6)³ of the fine-grid dimension, as in the one-block focusing method. The impact upon efficiency of the use of buffering space to maintain enough accuracy was also studied. It was found that an “optimal” multi-block dimension exists for a given computer hardware setup, and this dimension is more or less independent of the solute geometries. A parallel version of thedistributive focusing method was also implemented. Given the proper settings, the distributive method was able to achieve respectable parallel efficiency with tested biomolecular systems on a loosely connected computer cluster.     

Hunting Bambi—evaluating the basis for selective harvesting of juveniles

Human harvesting is often a major mortality factor and, hence, an important proximate factor driving the population dynamics of large mammals. Several selective harvesting regimes focus on removing animals with low reproductive value, such as “antlered” harvests in North America and juvenile harvesting in many European countries. Despite its widespread use and assumed impact, the scientific basis of juvenile harvesting is scattered in the literature and not empirically well-documented. We give the first overview of demographic, evolutionary and practical management arguments for selective harvesting of juveniles. Furthermore, we empirically test two demographic arguments based on harvest statistics of Red Deer (Cervus elaphus) in seven European countries. P₁: Harvesting juveniles has little influence on harvest growth compared with harvesting adult females due to the lower reproductive value of juveniles than adult females; P₂: Harvesting of juveniles dampens variance in harvest due to lower and more variable natural survival rates of juveniles compared with adults. We found that harvesting juveniles has little effect on harvest growth rate, while harvesting adult females has a significant negative effect (consistent with P₁), but that increasing the proportion of juveniles in the harvest did not decrease the variability in harvest between years (P₂ not supported). Based on our empirical findings and overview of arguments, we discuss how the merits of juvenile harvesting may vary over time as populations move from a low density to a very high density state.     

Is there a physical chemical basis for the present genetic code?

Summary Recent experiments on the interaction of amino acids with homopolyribonucleic acids as measured by proton magnetic resonance spectroscopy and on the interactions of ribonucleoside 5-monophosphates with immobilized amino acids give relative binding strengths that are not reconcilable with the present genetic code in any simple way.